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General Considerations for Acquiring a Three-Color Image by Laser Scanning Confocal Microscopy

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Confocal Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2304))

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Abstract

Laser scanning confocal microscopy is the workhorse epifluorescence imaging technique used in laboratories worldwide to acquire three-dimensional images of both fixed and live specimens with fine, high-contrast optical sections to discern details that cannot be afforded by standard widefield microscopy. This basic protocol steps the user through a typical three-color imaging experiment using a Zeiss LSM 880 confocal microscope for the example. The extensive Notes section attempts to generalize the method so that concepts and considerations can be applied to other laser scanning confocal systems.

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Acknowledgments

I would like to warmly thank Drs. Alama Arnold and James McIlvain for their patience and humor while answering all my Zeiss-related questions, my long-time colleague and friend Dr. Haewon Sohn, and my recent (and awesome) trainees: Jack Herrick, Meha Patel, and Jina Yom for critically reading this manuscript. This work is supported by intramural funding from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health.

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Correspondence to Joseph Brzostowski .

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© 2021 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply and Springer Nature US

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Brzostowski, J. (2021). General Considerations for Acquiring a Three-Color Image by Laser Scanning Confocal Microscopy. In: Brzostowski, J., Sohn, H. (eds) Confocal Microscopy. Methods in Molecular Biology, vol 2304. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1402-0_3

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  • DOI: https://doi.org/10.1007/978-1-0716-1402-0_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1401-3

  • Online ISBN: 978-1-0716-1402-0

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